Copper ions in carbomycin a fermentation

ABSTRACT

Addition of copper ions to carboymycin A-producing fermentation media gives rise to an increased yield of the antibiotic.

United States Patent Sardinas 1 Apr. 25, 1972 [54] COPPER IONS INCARBOMYCIN A [56] References Clted FERMENTATION UNITED STATES PATENTS[72] Gales 3,483,088 12/1969 Seeley ..195/1 14 [73] Assignee: Chu.Pfizer & Co., Inc., New York, N.Y. 2,796,379 6/1957 Tanner et al..l95/80 X [22] Filed: 6! 1969 Primary Examiner-Joseph M. Golian [21]AppL NOJ 805,018 Attorney-Connolly and Hutz [57] ABSTRACT Ad dition ofcqpperjons to c arboymycin A-producing ffarl'nen. Field 1 14 1 l7 tat1onmecha g1ves use to an mcreased y1eld of the ant1b1ot1c.

4 Claims, No Drawings COPPER IONS IN CARBOMYCIN A F ERMENTATIONBACKGROUND OF THE INVENTION This invention relates to the production ofthe antibiotic called carbomycin A, and, in particular to the use ofcopper ions in its production by fermentation.

Carbomycin A is an antibiotic with important antibacterial propertiesand wide clinical application in the treatment of bacterial infections.The use of an antibiotic, particularly on a large scale, requires thatit can be cheaply produced in large quantities.

in general terms, the media used in microbial fermentations include asource of nitrogen, a source of carbon, and nutrient salts. It is knownthat animals and microorganisms require certain trace elements as growthfactors, i.e., materials which are necessary for'growth and in theabsence of which growth is inhibited or impossible. Trace elements arethose elements, usually metals, which, although their physiological roleis oftentimes obscure, are essential for the growth of animals andmicroorganisms. These elements are usually present in minute or traceamounts in the microorganism and in its environment. Examples of some ofthese trace elements, all or some of which may be required as anecessary growth factor for a particular microorganism, are: cobalt,calcium, boron, zinc, manganese, sodium, potassium, and iron.

In the case of microbial fermentations, trace elements play an importantrole not only in cellular production but additionally the products ofmetabolism arising out of the growth of the microorganism. Theproduction of antibiotics, and in this instance, carbomycin A, isdependent on the special interplay of microbial growth and metabolicprocesses.

In the culture of antibiotic-producing microorganisms, the growth mediumusually contains in varying amounts those trace elements necessary forgrowthand antibiotic production. However, the amounts andtheavailability of the trace elements in the natural and complex substratematerials .varies with the particular source and, as a result,reproducible and satisfactory fermentation results are often difficultto obtain. It is therefore sometimes necessary to add additional amountsof one or more trace elements.

The locus of action of copper in the cell is poorly understood. Copperappears to be part of large protein molecules having a respiratoryfunction. The importance of copper as part of the trace element complexis well known to those skilled in the fermentation art. However, themarked stimulatory effect (20-30 percent) of copper ions on carbomycin Afermentation yields, beyond that obtainable with other trace elements,alone or in combination, is a surprising fermentation phenomenon.

SUMMARY OF THE INVENTION In general, this invention embodies a processfor increasing the carbomycin A fermentation yields by the addition ofcopper ions to the fermentationmedia.

DETAILED DESCRIPTION OF THE INVENTION A preferred process for theproduction of carbomycin A (U.S. Pat. No. 2,796,379 to F. W. Tanner,Jr., et al., June 18, 1957) involves the cultivation of Streptomycesnutrient medium ATTC-l 3449, preferably in an aqueous nutrient at atemperature of about 24 30 C., and under submerged conditions withagitation and aeration. Nutrient media which are useful for this processinclude a carbohydrate such as sugars, starch, glycerol, and cornstarch; a source of organic nitrogen such as that contained in casein,soybean meal, peanut meal, wheat gluten, cotton seed meal, lactalbumin,tryptone and enzymatic digest of casein. The use of enzymatic digest ofcasein as a nitrogen source is preferred. Additional growth substancesare provided by the addition of such substances as distillers solubles,yeast extract, molasses, or fermentation residues, as well as mineralsalts such as sodium chloride, potassium phosphate, sodium nitrate, andmagnesium sulfate.

A soluble, non-growth inhibiting nonionic (poly) oxyethylene glycolether surface-active agent, in an amount to provide a concentration ofabout 0.2-4 percent, is included in the fermentation; anti-foamingagents, such as vegetable oils, may be added to the fermentation medium.The pH of the fermentation tends to remain rather constant, but, ifvariations are encountered, a buffering agent such as calcium carbonatemay also be added to the medium.

An embodiment of this invention is the addition of a copper salt at sucha concentration that the final copper ion level is from about 40 to 65ppm, and preferably from about 50 to 55 ppm.

The copper ions are provided by copper salts such as cupric sulfate,cupric chloride, cupric acetate, cupric nitrate, cupric carbonate,cupric bromide, cuprous bromide, cuprous chloride, cuprous iodide andpreferably, cupric sulfate. The copper salt used is generally best addedat the time of medium make-up. However, it may be added as a sterilesolution or suspension up to 24 hours after inoculation of the medium.

inoculum for the preparation of antibiotic carbomycin A by the growth ofa strain of S. halstedii may be obtained by employing growth from slantsof such media as Emerson's agar or beef lactose. The growth may be usedto inoculate either shaken flasks or inoculum tanks for submergedgrowth, or, alternatively, the inoculum tanks may be seeded from theshaken flasks. The growth of the microorganism usually reaches itsmaximum in about 2 to 3 days. However, variations in the equipment used,the rate of aeration, rate of stirring, etc., may affect the speed withwhich the maximum activity is reached. In general, from about 24 hoursto 4 days is the desirable period for producing the antibiotic. Aerationof the medium in tanks for submerged growth is maintained at the rate ofabout one-half to two volumes of free air per volume of broth perminute. Agitation may be maintained by suitable types of agitatorsgenerally familiar to those in the fermentation industry. Asepticconditions, of course, must be maintained throughout the preparation andtransfer of the inoculum and the growth period of the microorganism.

Recovery of the antibiotic is accomplished by means generally familiarto those skilled in the art, such as extraction, precipitation and theuse of strong cation exchange resins.

The present invention embraces not only the use of the herein describedorganism but also of mutants thereof produced by subjecting the organismto such measures as X- radiation, ultraviolet radiation, nitrogenmustard and the like.

The following examples are merely illustrative and are not intended tolimit the invention, the scope of which is defined by the appendedclaims.

EXAMPLE I An inoculum is prepared, using a growth medium having thefollowing composition:

Grams/liter Cerelose 15.0 Soy flour 30.0 MgSO4'7H,O 1.0 Calciumcarbonate 10.0

The medium is adjusted to pH 6.8 with potassium hydroxide, and thensterilized and cooled. Spores of a 7-day old Emerson agar slant of S.halstedii ATCC-l3449 are transferred under aseptic conditions to 20 ml.of water, and a homogeneous suspension of spores is obtained by shaking.Six ml. of this suspension is transferred into 750 ml. of the abovemedium in a 2.8 liter Fernbach flask, and shaken for 48 hours at 30 C.

A nutrient medium is prepared having the following composition:

Gramslliter Enzose E-081 (sugar concentrate, Corn Products, Inc. 25.0ml. Beet molasses 22.5 Wheat starch 5.0 Cerelose l5.0

Casein Digest(5%) 400 ml.

The pH of the medium is adjusted to 7.0 with potassium EXAMPLE II Theprocess of Example I is repeated using 0.25 grams of CuSO,'5I-l,O perliter (Cu**, 55 ppm) in place of 0.20 grams per liter (Cu**, 50 ppm),with comparable results.

EXAMPLE III EXAMPLE IV The inoculum of Example I is used to inoculate anutrient medium having the following compositions:

Grams/liter Cerelose 25.0 Beet molasses l5.0 Wheat starch 5.0 Caseindigest (5%) 400 ml MnSO,-H,O 1.5 MgSO,'7H,O 1.25

CuSO '5I-I,0 I F020 (Cu**, 50 ppm) The pH of the medium is adjusted to7.0 with potassium hydroxide. One-half ml. Antifoam A (Dow ChemicalCompany) and '10 ml. Tergitol NP-35 (Union Carbide) are added per literof medium, and the mixture is sterilized.

A 10 percent aliquot of inoculum is transferred under aseptic conditionsinto the above nutrient medium. After agitation and aeration for 2 days,the broth potency is substantially greater than that of a controlfermentation without copper.

EXAMPLE V The process of Example IV is repeated using 0.25 grams of CuSO-5H O per liter (Cu*", 55 ppm) in place of 0.20 grams per liter (Cu**,50 ppm), with comparable results.

EXAMPLE VI The process of Example IV is repeated using 0.16 grams ofCuSO '5I-I;O per liter (Cu**', 40 ppm) in place of 0.20 grams per liter(Cu*", 50 ppm), with comparable results.

EXAMPLE VII comprises conducting the fermentation in the presence of acopper salt in an amount to provide a concentration of copper ions ofabout 40-65 ppm.

2. The process of claim 1 wherein said copper salt is cupric sulfate.

3. The process of claim 1 wherein said copper salt is selected from thegroup consisting of cupric chloride, cupric acetate, cupric nitrate,cupric carbonate, cupric bromide, cuprous bromide, cuprous chloride,cuprous iodide.

4. In the process for the production of carbomycin A by the cultivationof Streptomyces halstedii ATCC-13449 in an aqueous, nutrient mediumunder submerged, aerobic conditions, the improvement which comprisesconducting the fermentation in the presence of a copper salt in anamount to provide a concentration of copper ions of about 50-55 ppm.

2. The process of claim 1 wherein said copper Salt is cupric sulfate. 3.The process of claim 1 wherein said copper salt is selected from thegroup consisting of cupric chloride, cupric acetate, cupric nitrate,cupric carbonate, cupric bromide, cuprous bromide, cuprous chloride,cuprous iodide.
 4. In the process for the production of carbomycin A bythe cultivation of Streptomyces halstedii ATCC-13449 in an aqueous,nutrient medium under submerged, aerobic conditions, the improvementwhich comprises conducting the fermentation in the presence of a coppersalt in an amount to provide a concentration of copper ions of about50-55 ppm.